B. mallei and B. pseudomallei ("B. (pseudo)mallei"), two closely related Gram-negative bacteria, are serious potential bioterrorist agents listed on CDC/MMWR's category B list. These organisms cause life-threatening infections (Glanders' disease and Melioidosis, respectively), where antibiotic treatment is sometimes insufficient. The virulence factors of the two species are not well understood, and it is difficult to distinguish these species from each other and also from the closely related avirulent species B. thailandensis. B.(pseudo)mallei is also a civilian problem causing septicemia in Southeast Asia. Another Burkholderia species, B. cepacia, is a more common civilian problem that increases mortality and morbidity of cystic fibrosis patients. To better treat and diagnose B.(pseudo)mallei, we will develop monoclonal antibodies (mAbs) against potential virulence factors: components of the type III secretion system (TTSS). The presence of TTSS has been correlated to virulence of Burkholderia. Also, since several other invasive Gram-negative bacteria utilize TTSS to enter host cells, it is plausible that TTSS are critical for Burkholderia's virulence as well. TTSS components will be cloned and expressed in E.coli to generate sufficient quantities of purified antigen to generate mAbs. Fully human mAbs will be generated by phage display technology. Initial screening of the mAbs for reactivity will be conducted against a panel of Burkholderia isolates cultivated under different conditions, mAbs of interest will be further evaluated for protective capability in both in vitro and subsequently in vivo small animal models. We will also investigate potential differences of TTSS genes among isolates of Burkholderia. In summary the primary objectives of the project are: -Development of mAbs with utility in diagnosing and treating B.(pseudo)mallei and other Burkholderia species, including B.cepacia. -Improved understanding of the pathogenicity of B.(pseudo)mallei as a basis for potential vaccine development.